Post-transcriptional RNA processing can regulate gene expression, which is essential for the control of cellular metabolism, growth, and differentiation. Studying these processes in complex systems is often challenging and sometimes not even feasible. Archaea often have eukaryote-like processes, but at a basic level, thus serving as much simpler model systems to gain insights into these complex cellular events. Broad long-term objectives of this application are to characterize various RNA processing events in Archaea.
Specific aims of this proposal are: (1) Determination of the changes occurring in box C/D guide and target RNAs during assembly of sRNPs and during and after modification reactions;(2) In vivo characterization of the structure and function of archaeal Cbf5 protein and a guide H/ACA RNA;and (3) Identification of the determinants for tRNA Y54 synthase activity of Pus10 proteins. A variety of in vitro and in vivo techniques will be used to address the above-metioned aims. Lead(II) induced cleavage mapping of 32P-end-labeled T7 RNA polymerase generated transcripts will be done in presence of recombinant box C/D RNP core proteins for the structural studies of guide and target RNAs and their interactions during catalysis. To study the structural and functional significance of Cbf5 protein and H/ACA RNA, corresponding genes will be deleted in H. volcanii. The resulting deletion strains will be transformed with plasmid-borne copies of genes expressing mutant Cbf5 protein/HACA RNA, and the status of rRNA pseudouridylation will be checked using CMCT and U- specific reactions. Several mutant versions of Pus10 will be studied in vitro for their ability to produce pseudouridine at positions 54 and/or 55 of tRNAs. The effects of these mutants will be further studied using an E. coli based heterologous in vivo system. Defects in both box C/D and H/ACA sRNP components have been implicated in several diseases. Thus, the overall understanding of these processes in archaeal systems will help us understand similar processes in human systems under normal conditions, and the changes that may occur under diseased conditions.
Studies of gene regulatory phenomena such as RNA processing help us in understanding cellular metabolism and growth under normal as well as diseased conditions. Studying these processes in complex system is often challenging and sometimes not even feasible. In this proposal by using simple model organisms, we elucidate the mechanisms and functions of these events, which can then be correlated to human systems.